9-cis-retinoic acid inhibits androgen receptor activity through activation of retinoid X receptor

Although the retinoic X receptor (RXR) forms heterodimers with many members of the estrogen receptor subfamily, the interaction between RXR and the members of the glucocorticoid receptor subfamily remains unclear. Here we show that the RXR can form a heterodimer with the androgen receptor (AR) under in vitro and in vivo conditions. Functional analyses further demonstrated that the AR, in the presence or absence of androgen, can function as a repressor to suppress RXR target genes, thereby preventing the RXR binding to the RXR DNA response element. In contrast, RXR can function as a repressor to suppress AR target genes in the presence of 9-cis-retinoic acid, but unliganded RXR can function as a weak coactivator to moderately enhance AR transactivation. Together, these results not only reveal a unique interaction between members of the two nuclear receptor subfamilies, but also represent the first evidence showing a nuclear receptor (RXR) may function as either a repressor or a coactivator based on the ligand binding status.     

PDGF receptor activation induces p120-catenin phosphorylation at serine 879 via a PKCalpha-dependent pathway

p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell-cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCalpha, a conventional PKC isoform implicated previously in disruption of adherens junctions.     

Liver X receptor activation induces podocyte injury via inhibiting autophagic activity

Podocyte injury plays a key role in the occurrence and development of kidney diseases. Decreased autophagic activity in podocyte is closely related to its injury and the occurrence of proteinuria. Liver X receptors (LXRs), as metabolic nuclear receptors, participate in multiple pathophysiological processes and express in several tissues, including podocytes. Although the functional roles of LXRs in the liver, adipose tissue and intestine are well established; however, the effect of LXRs on podocytes function remains unclear. In this study, we used mouse podocytes cell line to investigate the effects of LXR activation on podocytes autophagy level and related signaling pathway by performing Western blotting, RT-PCR, GFP-mRFP-LC3 transfection, and immunofluorescence staining. Then, we tested this effect in STZ-induced diabetic mice. Transmission electron microscopy and immunohistochemistry were employed to explore the effects of LXR activation on podocytes function and autophagic activity. We found that LXR activation could inhibit autophagic flux through blocking the formation of autophagosome in podocytes in vitro which was possibly achieved by affecting AMPK, mTOR, and SIRT1 signaling pathways. Furthermore, LXR activation in vivo induced autophagy suppression in glomeruli, leading to aggravated podocyte injury. In summary, our findings indicated that activation of LXRs induced autophagy suppression, which in turn contributed to the podocyte injury.

     

Fluorescent retinoid X receptor ligands for fluorescence polarization assay

Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization (FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino]nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should be a lead compound for use in high-throughput assay systems for screening RXR ligands.     

Involvement of retinoid X receptor alpha in coenzyme Q metabolism

The nuclear retinoid X receptor alpha (RXRalpha) is the heterodimer partner in several nuclear receptors, some of them regulating lipid biosynthesis. Since coenzyme Q (CoQ) levels are greatly modified in aging and a number of diseases, we have investigated the involvement of RXRalpha in the biosynthetic regulation of this lipid by using a hepatocyte-specific RXRalpha-deficient mouse strain (RXRalpha-def). In the receptor-deficient liver, the amount of CoQ decreased to half of the control, and it was demonstrated that this decrease was caused by a significantly lowered rate of biosynthesis. On the other hand, induction of CoQ was extensive in both control and RXRalpha-def liver using the peroxisomal inducer di(2-ethylhexyl)phthalate (DEHP). Since the RXRalpha deficiency was specific to liver, no change in CoQ content or biosynthesis was observed in kidney. The other mevalonate pathway lipids, cholesterol and dolichol, were unchanged in the RXRalpha-def liver. Upon treatment with DEHP, cholesterol decreased in the control but remained unchanged in the receptor-deficient mice. In control mice, cold exposure elevated CoQ levels by 60%, but this induction did not occur in the liver of RXRalpha-def mice. In contrast, PPARalpha-null mice, which lack induction upon treatment with peroxisomal inducers, respond to cold exposure and CoQ content is increased. The amount of cholesterol decreased in both control and RXRalpha-def liver upon cold treatment. The results demonstrate that RXRalpha is required for CoQ biosynthesis and for its induction upon cold treatment, but does not appear to be involved in the basic synthesis of cholesterol and dolichol. The receptor is not involved in the elevated CoQ biosynthesis during peroxisomal induction.     

B cell receptor cross-talk: exposure to lipopolysaccharide induces an alternate pathway for B cell receptor-induced ERK phosphorylation and NF-kappa B activation

BCR signaling in naive B cells depends on the function of signalosome mediators; however, prior engagement of CD40 or of IL-4R produces an alternate signaling pathway in which Bruton's tyrosine kinase, PI3K, phospholipase Cgamma2, and protein kinase Cbeta are no longer required for BCR-induced downstream events. To explore the range of mediators capable of producing such an alternate pathway for BCR signaling, we examined the TLR4 agonist, LPS. B cell treatment with LPS at relatively low doses altered subsequent BCR signaling such that ERK phosphorylation and NF-kappaB activation occurred in a PI3K-independent manner. This effect of LPS extended to MEK phosphorylation and IkappaBalpha degradation, and it developed slowly over a period of 16-24 h. The involvement of TLRs is suggested by similar effects observed with a structurally distinct TLR agonist, PAM3CSK4 and by the need for MyD88 for induction of alternate BCR signaling by LPS. Thus, LPS-mediated TLR engagement produces an alternate pathway for BCR-triggered signal propagation that differs from the classical, signalosome-dependent pathway.     

Evidence for a novel cardiac-enriched retinoid X receptor partner.

Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.     

Mutations in retinoid X receptor that impair heterodimerization with specific nuclear hormone receptor

Retinoid X receptor (RXR) serves as a promiscuous heterodimerization partner for many nuclear receptors through the identity box, a 40-amino acid subregion within the ligand binding domain. In this study, we randomly mutated two specific residues within the human RXRalpha identity box region previously identified as important determinants in heterodimerization (i.e. Ala(416) and Arg(421)). Interestingly, most of these mutants still retained wild type interactions with thyroid hormone receptor (TR), retinoic acid receptor, peroxisome proliferator-activated receptor alpha, small heterodimer partner, and constitutive androstane receptor. However, RXR-A416D and R421L were specifically impaired for interactions with TR, whereas RXR-A416K lost both TR and retinoic acid receptor interactions. Accordingly, RXR-A416D did not support T3 transactivation in mammalian cells, whereas RXR-A416K was not supportive of transactivation by retinoids or T3. These results provide a basis upon which to further design mutant RXRs highly selective in heterodimerization, potentially useful tools to probe nuclear receptor function in vivo.